Alphabeta generation occurs via serial cleavage of APP by p- and gamma-secretase, but is precluded via serial cleavage by alpha- and gamma-secretase. Project 1 (Selkoe/Wolfe) has generated evidence for an alpha/gamma-secretase complex, which we have termed the sheddasome. In this project, we will explore factors that can modulate the sheddasome either pharmacologically (using novel gamma-secretase modulators [GSM]), or genetically (using novel late-onset Alzheimer's disease [LOAD] mutations in ADAMVO which we discovered during the current PPG period). In our first set of aims, we will carry out studies of a novel series of highly potent, APP-specific GSMs. These GSMs are aryl 2-aminothiazole GSMs that bind directly to the gamma-secretase complex, decreasing AP42 and AP40 levels and increasing APas and AP37 levels. This project will serve to leverage an independent ongoing project on these GSMs supported by the NIH Blueprint Neurotherapeutics Network (NIH-BNN). While we receive no funding from the NIH-BNN, we co-developed these GSM' and serve as close collaborators and Lead Development Team members for the project. As part of Aim 1, in collaboration with Project 1, we will test for the effects of these novel GSMs on gamma-secretase preparations, including gamma-secretase- enriched membranes and sheddasome complexes. We will also test whether the GSMs allosterically impact PS1 conformation in the gamma-secretase complex in collaboration with Project 3 (Berezovska/Hyman). Finally, we will test for the potency and substrate selectivity of these GSMs in collaboration with Project 4 (Kovacs). In the second set of aims, we will characterize two rare LOAD missense mutations in the prodomain oi ADAMIO, which we have already shown to tightly co-segregate with LOAD in 7 families (age of onset ~70 yr). We have also shown that both of these mutations significantly attenuate alpha-secretase activity and elevate alphabeta levels (relative to wild-type) in vitro and in vivo. We have already generated transgenic mice overexpressing either wild-type (WT) or mutant (Q170H; R181G; dominant-negative) forms of ADAMIO. We will use these animal models to identify physiologic ADAM 10 substrates, and characterize their serial cleavage by gamma-secretase (with Project 4). In collaboration with Project 1, we will test whether the LOAD and dominant negative mutations in ADAMIO affect the interaction of ADAM10 with gamma-secretase in the sheddasome.